Noninvasive Detection of HER2 Expression in Gastric Cancer by 64Cu

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Non-invasive Detection of HER2 Expression in Gastric Cancer by 64Cu-NOTA-Trastuzumab in PDX Mouse Model and in Patients Xiaoyi Guo, Hua Zhu, Nina Zhou, Zuhua Chen, Teli Liu, Fei Liu, Xiaoxia Xu, Hongjun Jin, Lin Shen, Jing Gao, and Zhi Yang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00673 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018

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Molecular Pharmaceutics

Non-invasive Detection of HER2 Expression in Gastric Cancer by 64

Cu-NOTA-Trastuzumab in PDX Mouse Model and in Patients

Xiaoyi Guo1, †, Hua Zhu1, †, Nina Zhou1, Zuhua Chen2, Teli Liu1, Fei Liu1, Xiaoxia Xu1, Hongjun Jin3, Lin Shen2, Jing Gao2,*, Zhi Yang1,* 1.

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing),

Department of Nuclear Medicine; Peking University Cancer Hospital & Institute, Beijing, 100142, China 2.

Department of Gastrointestinal oncology, Peking University Cancer Hospital & Institute, Beijing

100142, China; 3.

Research Center of Molecular Imaging and Engineering, Sun Yat-sen University, the Fifth Affiliation

Hospital, Zhuhai, Guangdong Province，519000，China.

†

These authors contributed equally in this work

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ABSTRACT: The purpose of this study was to establish the quality control, and quantify the novel 64

Cu-NOTA-Trastuzumab in gastric cancer patient-derived xenografts (PDX) mice models and patients

by applying the molecular imaging technique. Trastuzumab was labeled with

64

Cu using

NCS-Bz-NOTA as bi-functional chelator, and hIgG1 was labeled with same procedures as a negative control agent. HER2-positive (case 176, n = 12) and HER2-negative (case 168, n=3) PDX models were established and validated by western blot, DNA amplification and immunohistochemistry (IHC). Both models 64

were

conducted

Cu-NOTA-Trastuzumab or

for 64

micro-PET

imaging

by

tail

injection

of

18.5

MBq

of

Cu-NOTA-hIgG1. Radio-probes uptake in tumor and main organs were

quantified by region of interested (ROI) analysis of the micro-PET images and autoradiography. Finally, gastric cancer patients enrolled in the preliminary

64

Cu-NOTA-Trastuzumab PET/CT scans.

NOTA-Trastuzumab was efficiently radiolabeled with 64Cu over a 99% radio-chemical purity and 17.5 GBq/µmol specific activity. The immune-activity were preserved as the non-modified antibody, and the radiopharmaceutical proved to be stable for up to 5 half-decay life of 64Cu both in-vitro and in-vivo. Two serials of PDX gastric cancer models were successfully established: case 176 for HER2 positive and case 168 for HER2 negative. In micro-PET imaging studies, 64

Cu-NOTA-Trastuzumab exhibits a significant higher tumor uptake (11.45 ± 0.42 ID%/g)

compared with 64Cu-NOTA-IgG1 (3.25 ± 0.28 ID%/g, n = 5, p = 0.0004) at 36h after intravenous injection. Lower level uptake of 64Cu-NOTA-Trastuzumab (6.35 ± 0.48 ID%/g) in HER2-negative PDX tumor models further confirmed specific binding of the radio-probe. Interestingly, the co-injection of 2.0 mg Trastuzumab (15.52 ± 1.97 ID%/g) or 2.0 mg hIgG1 (15.64 ± 3.54 ID%/g) increased the with

64

64

Cu-NOTA-Trastuzumab tumor uptake in PDX tumor (HER2+) models compared

Cu-NOTA-Trastuzumab alone (p < 0.05) at 36h post injection. There were good 2 / 35


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correlations between micro-PET images and IHC (n = 4) and autoradiography in PDX (HER2+) tumor tissues. Therefore, 64

64

Cu-NOTA-Trastuzumab successfully translated to clinical PET imaging,

Cu-NOTA-Trastuzumab PET/CT scan in gastric cancer patients showed good detection ability. In

conclusion, we reported quality control and application of novel

64

Cu-NOTA-Trastuzumab for HER2

expression in PDX gastric cancer mice models and gastric cancer patients. Moreover, 64

Cu-NOTA-Trastuzumab holds great potential for non-invasive PET detection, staging, and follow-up

of HER2 expression in gastric cancer. KEYWORDS: Patient-Derived Xenograft model, 64Cu-NOTA-Trastuzumab, HER2, Gastric Cancer

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INTRODUCTION Human epidermal receptor type 2 (HER2; also known as ErbB2) is one of the members of HER family which is encoded by the HER2 gene (also known as HER2/neu and ErbB2 gene). The HER2 pathway promotes cell growth and division when it is functioning normally; however, when it is overexpressed, cell growth accelerates beyond its normal limits. In cancer cells, the HER2 protein can be expressed up to 100 times more than in normal cells (2,000,000 versus 20,000 per cell)1-4. This overexpression leads to strong and constantly proliferative signaling and hence tumor formation. Overexpression of HER2 also causes deactivation of checkpoints, allowing for even greater increases in proliferation5-7. Trastuzumab is an engineered humanized IgG1 monoclonal antibody (mAb) recognizing an epitope in the extracellular domain of the receptor and is used for immunotherapy for HER2/neu-positive cancers. Trastuzumab was fast-tracked by the Food and Drug Administration (FDA) and gained approval in September 1998. Recently, trastuzumab has been demonstrated to increase overall survival in HER2 overexpression locally advanced or metastatic gastric cancer. HER2 is over-expressed in 7-34% of patients with gastric cancer. High tumor heterogeneity can exist between 8

primary tumors and the metastases, between different metastases . In addition, HER2 expression can vary during the course of the treatment. Accurate characterization of HER2 expression is essential for optimal therapy. In routine clinical laboratory, the most commonly employed methods are immunohistochemistry(IHC) and fluorescent in situ hybridization (FISH). These ex vivo methods are local and invasive. Furthermore, discordance of primary tumors and metastases are difficult to be measured by IHC/FISH. There exists a compelling need accurately detected the whole body HER2 expression in patients in order to guide the trastuzumab based treatment. Full body non-invasive positron emission tomography (PET) imaging of HER2 expression in advanced gastric cancer provides 4 / 35


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precisely qualitative and quantitative information of all lesions, including primary and non-accessible metastases. Lots of PET radionuclides labeled trastuzumab or pertuzumab have been proven to be effective for HER2-positive lesions detection9-17. Paradoxically, these individual probes, designed and synthesized to enable “precision medicine,” evaluated in a commercial cell line based tumor models. And almost all cell lines were sub-cultured many circles and may lost most of patient’s original characteristics18. More seriously, the microenvironments of these tumor models were completely different from patient’s in-suit tumors due to lack of blood vessel supply, tumor-associated stroma and so on19. Considering the high heterogeneity of gastric cancer, patient-derived tumor xenograft (PDX) tumor models are regarded as superior to traditional cell derivatives xenografts (CDX) models, as both the microenvironments and individual characters are important for tumor metastases and therapy monitoring. We previously reported the generation of gastric cancer based PDX models using gastroscopic biopsies technology20. PDX gastric tumor models have been confirmed to maintain the similar biological characteristics of the parental tumors. And we confirmed the potential for using PDXs to investigate the in-vivo/vitro antitumor activity of dual PI3K/mTOR inhibitor BEZ235 in HER2-positive gastric cancer21. Here, we report the quality control, metabolism analysis and micro-PET evaluation of novel 64Cu-NOTA-Trastuzumab, and applied 64

this

probe

in

gastric

cancer

patient-derived

xenografts

models.

What’s

more,

Cu-NOTA-Trastuzumab was successfully applied in gastric cancer patient PET clinical translational

study. Those results showed that novel

64

Cu-NOTA-Trastuzumab holds great potential for detecting

HER2 levels noninvasively.

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MATERIALS AND METHODS Reagents,

64

Cu Radiolabeling Antibody and Quality Control. Details of the reagents and cell

culture are provided in the supplemental materials. Radioactive

64

CuCl2 was supplied by the

Department of Nuclear Medicine, Peking University Cancer Hospital & Institute in Beijing as 3.7 22

MBq/µL in 0.01 M HCl solution . Trastuzumab or non-specific hIgG1 antibody were conjugated with NCS-Bz-NOTA at a molar ratio of 1 : 3 at pH 8.4 for 4h as we previously reported23. Stability and Metabolism Analysis of Labeled Antibody. The stability of the labeled antibody was evaluated at 4oC in saline and 37oC in 5% human serum album for 60 h. The radiochemical purity 23

and specific activity were determined by Radio-TLC as we previously described . These data are shown in supplement materials. Patient-Derived Xenograft Models in NOD/SCID Mice. All animal experiments were performed in accordance with guidelines of the Animal Care and Use Committee of Peking University (Ethics Approval License: 2015KT08). HER2-positive (n = 12) and HER2-negatvie (n = 3) patient-derived xenografts (PDX) models were built for micro-PET imaging. HER2 expression level and the characteristics of these two patients are shown Table 1 and Figure 1 and Figure S3, respectively. When the tumor volume reached 0.5-1.0 cm in diameter, mice were randomly assigned. Bio-distribution and Micro-PET Imaging of

64

Cu-NOTA-Trastuzumab in Mice Models.

Details of bio-distribution can be found in supplemental materials. Micro-PET images were conducted with

18

F-FDG,

64

Cu-NOTA-Trastuzumab, or

64

Cu-NOTA-hIgG1 on dedicated micro-PET/CT

equipment (Super-Argus, SEDECAL, Spain) that had a field of view of 12.0 cm. Details of scan and reconstruction information can be found in supplemental materials. Immunohistochemistry and Autoradiography Analysis of PDX Tumor Tissues. PDX tumor 6 / 35


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models HER2/neu expression were confirmed by both H&E staining and IHC. Formalin-fixed, paraffin-embedded tumors were stained with antibodies against HER2/neu (HercepTest; DAKO). The autoradiography analysis was performed as previously reported20. Details of IHC and autoradiography analysis information can be found in supplemental materials. Gastric Cancer Patient Enrollment and Clinical PET/CT imaging. Gastric cancer patients (pathologically confirmed HER2 positive (IHC 3+ or FISH-positive)) were recruited from Peking University Cancer Hospital & Institute (The main exclusion criteria were severely liver or kidney dysfunction, pregnant or lactating, or those who couldn’t keep lying in the scan bed for more than 1h). Patients received detailed informed consents before the PET/CT examination. The Peking University Cancer Hospital & Institute Ethics Committee (Permission No. 2018KT02) approved the study. One gastric cancer patient (61, female), based on the pathological analysis, was diagnosed as middle differentiated papillary adenocarcinoma in the gastric antrum. The IHC illustrated EBER(-), EGFR(+), HER(3+), MLH1(+), MSH2(+), MSH6(+), PD-L1(+5%) and PMS2(+). The FISH test showed Her2 : CEP17 signal ratio ≥ 2.0. She had multiple liver metastases and planned to trastuzumab target therapy, agreed to participate in this study. Patient undergone 18F-FDG PET/CT was conducted at 3 days before 64Cu-NOTA-Trastuzumab PET/CT imaging studies. 185 MBq 64Cu-NOTA-Trastuzumab was injected intravenously along with 10 mg cold trastuzumab. PET/CT scans performed at 1h after 18

F-FDG injection and 1, 24, 48h after

64

Cu-NOTA-Trastuzumab injection with Siemens Biograph

mCT Flow 64 scanner (Erlangen, Germany). A Siemens workstation (MultiModality Workplace) was used for data processing. Two experienced nuclear medical doctors read the PET/CT images. Results were checked by two independent physicians. PET image evaluation and quantification of the maximum single-voxel standardized uptake value (SUVmax) was measured. 7 / 35



Statistical Analysis. The in vitro percentage of injected radio probe dose per gram of tissue presented as the mean ± standard deviation. ROI-based quantification micro-PET analysis data were analyzed using two-tailed, unpaired Student t-test, with p values less than 0.05 considered statistically significant. These statistical computations were performed using the Excel software program (Microsoft Corporation, Redmond, WA) or Graph Pad software (Graph Pad prism 5).

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Page 8 of 36

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RESULTS ELISA Analysis. The immune-activity of NOTA-Trastuzumab and the control agent NOTA-hIgG1 were tested by ELISA. As shown in Figure 1A, NOTA-trastuzumab almost reserved 99% immuno-activity compared with non-modified trastuzumab, while NOTA-hIgG1 had only background level binding to HER2. The high binding affinity of NOTA-trastuzumab might be due to only one NOTA chelator attached in each antibody. The number of NOTA chelating groups per antibody was estimated to be 1.0 by MALDI-TOF (Figure S2A), the M/Z = 148956.16 for NOTA-trastuzumab compared with M/Z = 148325.168 for trastuzumab (date not shown). HER2 Expression Analysis of PDX-tumor Mice Models. The HER2 expression of those two serials of PDX tumors were confirmed by both DNA amplification analysis (Figure 1 B) and western blot (Figure 1 C). Results showed the average DNA copy number of case 176 over 15000 while that number of case 168 less than 100. This was consistent with the western blot analysis. The H&E staining of each mouse tumor was showed in Figure 1D, and HER2 expression in PDX models tumors were confirmed by IHC and were shown in Figure 1E. It was clear that the case 168 based tumor tissues had low HER2 expression while the case 176 exhibited high HER2 expression. The schematic procedure for generating PDX mice models was shown in Figure S3. The biological characteristics of the PDX models were high consistent with gastric cancer patients, which supposed to be a superb tumor model for preclinical and clinical translation studies.

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Figure 1. Validate the immune-reactivity for each conjugate and HER2+ clinical cases. A. ELISA analysis of the immune-activity for trastuzumab (red circle), NOTA-trastuzumab (blue squares) and NOTA-hIgG1(blue triangles); B. DNA amplification analysis using high-throughput sequencing technology of case 176 along with control; C. The protein expression of HER2 was quantified by western blot analysis; D./E. H&E staining (D) and IHC (E) analysis of HER2 expression in 2 serials of PDX tumor tissues. 64

Cu-NOTA-Trastuzumab

Production

and

Quality

Control.

The

synthesis

of

NOTA-Trastuzumab and 64Cu radiolabeling were performed under good-laboratory-practice conditions with quality control shown in Table S1. The radiolabeling yield for 64Cu-NOTA-trastuzumab was 80.6 ± 3.0% (n = 6), and the radiochemical purity after PD-10 column purification was over 99.0% (Figure S2B). The average specific activity of 64Cu-NOTA-Trastuzumab was 17.5 GBq/µmol (123.3 MBq/mg) (n = 6). Radio-TLC and HPLC analysis of the radiopharmaceuticals revealed no aggregates, fragments, or radioactive impurities. The radio probe solution was then diluted to approximately 92.5 MBq/mL with saline and was filtered with a 0.22-µm MillexLG filter (EMD Millipore). The production of 64

Cu-NOTA-trastuzumab proved to be sterile and endotoxin free. For animal studies, injection 0.2 ml 10 / 35


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(17.5 MBq) of 64Cu-NOTA-trastuzumab for each PDX mouse for micro-PET imaging study. 64

Cu-NOTA-trastuzumab appeared to be rather stable with mean decrease in radiochemical purity

of less than 1.0% in both saline at 4oC and 5% HSA at 37oC after 60h incubation. Decomposition or dissociation of the complexes to either

64

Cu or other side products were not observed under

experimental conditions. These results indicated that

64

Cu-NOTA-Trastuzumab had suitable in vitro

stability for potential clinical applications. Bio-distribution studies and Micro-PET Imaging of Each Radio-probe. As shown in Figure S2C, the bio-distribution of

64

Cu-NOTA-trastuzumab with or without co-injection antibodies in KM

mice showed similar organs distribution. That might be due to the similar blood clearance of each dose. The photographs of PDX gastric cancer mice models were shown in Figure 2 A, C, D (left side). These photos exhibited the irregular tumor tissues of 0.4-1.0 cm in diameter, and they were a bit different with the CDX globose tumors. The whole body CT scans confirmed the anatomy structure of PDX models as seen in Figure 2A (Middle).

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Figure 2. Micro-PET images of PDX models. Decay-corrected whole-body coronal micro-PET images of

18

F-FDG (A),

64

Cu-NOTA-trastuzumab(B),

64

Cu-NOTA-hIgG1(C) in HER2 positive PDX models

and 64Cu-NOTA-Trastuzumab in HER2 negative PDX models (D) at each time point in the same scale bar. Red arrow indicates tumor and yellow arrow indicates liver. The representative coronal PET images of HER2 positive (case 176) or HER2 negative (case 168) PDX models at each time point were shown in Figure 2, and the quantification analysis of PET images were displayed in Figure 4. 18F-FDG was selected as a standard radio-probe for PET imaging as shown in Figure 2 A (right side). Twenty-four hours after 64

18

F-FDG micro-PET/CT imaging,

Cu-NOTA-trastuzumab was injected, and it showed obviously high uptake at 12h to 17.54 ± 1.15

ID%/g in tumor tissue by the exact same mouse, and got better contrast imaging at 36h, as shown in Figure 2B. 64Cu-NOTA-hIgG1, as a negative control probe, exhibits low tumor uptake both at 12h to 6.02 ± 0.70 ID%/g in HER2 positive PDX models, and got no better accumulation at 36h in Figure 2C. Also, 64Cu-NOTA-trastuzumab in HER2 negative PDX models (case 168, Figure 2D) showed minimal tumor uptake compared with HER2 positive PDX models (case 176, Figure 2B) at each time point. As shown in Figure 2, there were obviously differences among tumor uptake in PDX models (both HER2 positive and negative) after intravenously injected each tracer.

64

Cu-NOTA-Trastuzumab

exhibited the highest tumor uptake in HER2 positive PDX models compared with 64

Cu-NOTA-hIgG1. At the same time,

64

18

F-FDG and

Cu-NOTA-Trastuzumab exhibited low uptake in HER2

negative PDX models. Altogether, Figure 2 exhibited the specificity of

64

Cu-NOTA-Trastuzumab for

the detection of HER2 over-expression tumors, and the feasibility of using PDX models for potential PET/CT clinical translational study. In the protein dose escalation study in Figure 3, 0.5 mg, 2.0 mg trastuzumab and 2.0 mg hIgG1 were co-injected with 18.5 MBq

64

Cu-NOTA-Trastuzumab. Micro-PET imaging revealed consistent

12 / 35


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and excellent tumor uptake in all HER2 positive PDX models (n = 12). The uptake by tumor demonstrated clearly at 12h after 64Cu-NOTA-trastuzumab injection, although blood-pool activity was dominant at first several hours. With the radio-pharmaceutical’s metabolism, tumor uptake visually increased, whereas blood-pool activity decreased. As shown in Figure 3, almost all dose of radiopharmaceuticals exhibited high tumor uptake while the other organs remain a relative low uptake at 36h p.i. of 64Cu-NOTA-Trastuzumab in the same scale bar.

Figure 3. Micro-PET imaging of 64Cu-NOTA-Trastuzumab with protein dose escalation of co-injection with cold trastuzumab. Micro-PET imaging of

64

Cu-NOTA-Trastuzumab alone (A) and protein dose

escalation study of co-injection with 0.5 mg trastuzumab (B); 2.0 mg hIgG1 (C) and 2.0 mg trastuzumab (D) in HER2 positive (case 176) NOD-SCID PDX mice models at each time point in the same scale bar. Red arrow indicates tumor and yellow arrow indicates liver.

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Page 14 of 36

Figure 4. Region of interest (ROI) quantification of the micro-PET studies. Region of interest (ROI) analysis of the micro-PET images at 12h (A) and 36h (B) p.i. of 64Cu-NOTA-Trastuaumab (64Cu-Tras.), 64

Cu-NOTA-Trastuaumab

co-injection

with

0.5

64

Cu-NOTA-Trastuaumab

co-injection

with

2.0

64

Cu-NOTA-Trastuaumab

co-injection

64

Cu-NOTA-hIgG1(64Cu-hIgG) uptake values in tumor, heart, liver, spleen and muscle; values are

with

2.0

mg

trastuzumab

mg mg

(0.5

mg

Tras.C.I.),

(2

mg

Tras.C.I.),

trastuzumab hIgG1

(2

mg

hIgG

C.I.),

and

presented as %ID/g ± SD (n = 3 or 4). C Comparison of micro-PET ROI-derived quantification of 64

Cu-NOTA-Trastuaumab vs

64

Cu-NOTA-hIgG1 uptake in tumor section.

(* indicates p

Noninvasive Detection of HER2 Expression in Gastric Cancer by 64Cu

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